Fig. 1
AF-MSC proliferation capacity, morphology, phenotype and immunosuppressive effect. Graph showing the growth of AF-MSCs (at P2) over a 10-day culture period A. Doubling time for AF-MSCs at different passages (from P2 to P5) B. AF-MSC morphology visualized by fluorescence microscopy for the identification of phalloidin (green) and the cell nucleus are stained using DAPI (blue) C. Images taken at 20 × magnification, scale bar: 10 µm. Quantitative PCR analysis for the expression of pluripotent (Nanog, Oct4) mesenchymal (cd73, cd90, cd44 and cd105) and hematopoietic (cd34 and cd45) markers in AF-MSCs at passage P2 D. Flow cytometric analysis revealing the percentage of AF-MSCs positive for MSC-associated markers (CD29, CD73, CD90 and CD44). Results are presented as percentage of marker-positive cells and as average of three biological replicates ± standard deviation E. Expression of immunosuppressive markers in AF-MSCs as revealed by qPCR following 24 and 48 h treatment with a cocktail of pro-inflammatory cytokines (TNF-α and IFN-g, 20 ng/ml) F. Data represent the average of fold as fold-change compared with the expression levels found in the untreated cells (* = p > 0.05; ** = p < 0.01). Immunosuppressive effect of AF-MSCs on monocyte proliferation (ThP-1 cell line) as shown by flow cytometry G. Three different culture conditions were set: 1) Cell-to-cell contact, 2) Conditioned Media (CM), and 3) Transwell system. Resting and PHA-activated ThP-1 cells (aThP-1) were included in the study as negative and positive controls, respectively. Data represent the percentage ± SD of three biological replicates (* = p > 0.05; ** = p < 0.01; *** = p < 0.001). H qPCR analysis of anti(Il-10, Tgf-b, Ccl22, Mrc1)- and pro(iNos, IL-6, Tnf-a)- inflammatory gene expression in undifferentiated macrophages (MF) or inflamed macrophages (MF1), respectively, exposed to CM from AF-MSC grown in standard conditions (AF-MSCs) or primed with pro-inflammatory cytokines (iAF-MSCs). Data are normalized to housekeeping gene expression (Gapdh) and presented as fold change relative to MF not exposed to CM for anti-inflammatory genes and to MF1 for pro-inflammatory markers (n = 3). Error bars represent the mean ± SD; ** = p > 0.05; ** = p < 0.01)